1. Field of the Invention
The invention relates to the field of immunoassay techniques for the identification and quantification of antigens, haptens, and antibodies and other compounds and substances of interest in biological fluids, and to the subfields of enzyme-immunoassay techniques and general methods for increasing specific activity of marker units.
2. Description of Prior Art
There are several types of test systems useful for the detection and measurement in serum or other media of biologically important or interesting compounds or substances:
1. Radioassay techniques (RIA)
a. Competitive protein binding assays PA1 B. Radioimmunoassay PA1 C. Immunoradiometric assays PA1 D. Sandwich or 2-site immunoradiometric assays PA1 1. Available and inexpensive in high purity. PA1 2. High enzymatic specific activity. PA1 3. Soluble under labeling and assay conditions. PA1 4. Chemically and functionally stable under labeling and assay conditions. PA1 5. Enzymatic activity detected simply, sensitively, inexpensively, rapidly and with standard laboratory equipment. PA1 6. Missing or in negligible concentration in analysis. PA1 7. Interfering factors missing in analyte. PA1 Acridine Orange PA1 5-Amino-2,3-dihydro-1,4-phthalazinedione PA1 7-Amino-1,3-naphthalenedisulfonic Acid PA1 4-Amino-1-naphthalenesulfonic Acid PA1 p-Anisaldehyde PA1 Chromotropic Acid PA1 Coumarin PA1 2',7'-Dichlorofluorescein PA1 6,7-Dihydroxy-4-methylcoumarin PA1 Eosin PA1 Erythrosin PA1 Fluorescamine PA1 Fluorescein PA1 1-Hydroxy-2-naphthoic Acid PA1 3-Hydroxy-2-naphthoic Acid PA1 trans-o-Hydroxycinnamic Acid PA1 4-Methylumbelliferone PA1 Morin PA1 1-Naphthol PA1 2-Naphthol PA1 1-Naphthol-3,6-disulfonic Acid PA1 2-Naphthol-6,8-disulfonic Acid PA1 1-Naphthol-2-sulfonic Acid PA1 1-Naphthol-4-sulfonic Acid PA1 2-Naphthol-6-sulfonic Acid PA1 1-Naphthylamine PA1 o-Phenylenediamine PA1 p-Phenylenediamine PA1 Phloxine B PA1 Resourfin PA1 Rhodamine PA1 Salicylic Acid PA1 2',4',5',7'-Tetrabromofluorescein PA1 X-poly-1-lysine-(HRPO).sub.1 PA1 X-poly-1-lysine-(HRPO).sub.2 PA1 X-poly-1-lysine-(HRPO).sub.3 PA1 X-poly-1-lysine-(HRPO).sub.4 and so forth.
2. Fluoroimmunoassays (FIA)
3. Enzyme immunoassay (EIA)
4. Lysis-initiating immunoassays (LIA)
5. Latex-particle agglutination (LPA)
6. Charcoal-particle agglutination (CPA)
7. Hemagglutination and Hemagglutination Inhibition Assays (HA)
8. Complement Fixation (CF)
9. Counter-electrophoresis, Immunoelectrophoresis (CEP)
10. Radial Immunodiffusion and Double diffusion (RID)
11. Viroimmunoassay (VIA); and
12. Spin immunoassay (SIA) among others.
Most of these tests are limited by one or more of the following limitations: (1) lack of sensitivity, (2) complexity of the test procedure, (3) instability of reagents, (4) hazardous reagents, (5) impure reagents, and (6) expensive equipment required.
Of the limitations cited above, the most serious is lack of adequate sensitivity. In general three levels of sensitivity are recognizable. Low sensitivity techniques, where materials detected and measured exiin microgram/milliliter quantities, include RID, CF, CEP, CPA, and LPA. Intermediate sensitivity techniques, where microgram/milliliter to nanogram/milliliter quantities of materials may be measured, include HA, CF, FIA, SIA, VIA, and EIA. Until now only RIA was able to measure with ultrasensitivity the picogram/milliliter to femtogram/milliliter region.
Many of the techniques listed required that some form of physical or chemically identifiable label be attached to reagents in the assay system in order that the result of a test can be detected, RIA, FIA, EIA, LIA, VIA, and SIA all fall into this category. Radioactivity, fluorescent moieties, enzymes, complement, viruses, and electron-spin labels are used respectively to generate some form of end-point signal. The sensitivity with which these labels can be detected directly and fundamentally affects the useful ranges of the test systems using them. For a review of the development and evaluation of immunological methods and their uses as diagnostic laboratory tools, reference is made to "Immunology as a Laboratory Tool" by Frans Peeton, in American Journal of Medical Technology, 37, #12, 455-469 (1971).
The sensitivity with which a labelling moiety can be measured depends upon the nature of the signal it generates, the ability to detect that signal, and upon the intensity of signal available per unit amount of marker molecule--its specific activity. With radioactive labels, heretofore the most popular label in use, the signal is decay radiation. Because of the penetrating properties of the emissions generated, radioactive decay can be detected easily. Modern counting equipment very efficiently measures the radioactive emissions from even a small amount of radioactivity. Finally, there is a range of specific activities offered by isotopes currently used for tagging.
Table I ______________________________________ Some Properties of Radioactive Isotopes Widely Used for Tagging Biological Materials Specific Activity of Isotope Pure Isotope (Curies/Mole) Half Life ______________________________________ 14.sub.C 6.25 .times. 10.sup.1 5720 years 3.sub.H 2.91 .times. 10.sup.4 12.3 years 35.sub.S 1.50 .times. 10.sup.6 87 days 125.sub.I 2.18 .times. 10.sup.6 60 days 32.sub.P 3.16 .times. 10.sup.6 14.3 days 131.sub.I 1.62 .times. 10.sup.7 8.1 days ______________________________________
The specific activity of a radioactively tagged compound depends upon several factors: the half life of the isotope used for labeling, the isotopic purity of the label, and finally how much of the label has been incorporated into the compound.
Radioactive isotopes of many elements are available for taggingbiological materials. Each isotope has its own unique half life. Thus a wide selection of specific activities for tagging are available merely byselecting an optimum radioisotope. Table I lists several commonly used radioisotopes, their specific activities and half lines. In general for use in immunoassays, the higher the specific activity of a radiolabeled compound, the better.
In a similar way enzymes may be characterized by the rate of catalytic effects on their respective substrates, their isoenzymatic purities, and the degree of incorporation of enzyme into labeled compound. In general however, if the enzyme is specified, the variables which can be adjusted to improve specific enzymatic activity are limited to the degree of incorporation into the tagged compound.
In contrast with radioiodine where several isotopes are available, selected isoenzymes of an enzyme family are often not available or are prohibitively expensive. The enzymes are specific to unique or narrowly restricted groups of substrates and thus substrate turn over rates, the rate at which enzyme catalyzes a substrate reaction, are fixed. Finally examples have been found which indicate that inclusion of several enzyme moieties per labeled molecule frequently destroys the characteristics for which that molecule was selected.
The thrust of this new invention is a way of incorporating two or more enzyme moieties (but not merely limited to enzymes) into a label without destroying the properties for which the labeled compound was originally selected. This is accomplished by introducing a novel linking moiety to the labeled compound. Attached to the linking moiety are the labeling enzymes in amounts that would normally alter the properties of the labeled compound if they were directly attached. The compound labeled in this way has greatly elevated specific enzymatic properties without losing the properties for which it was selected.
Up to the present time, the radioimmunoassay (RIA) method in its various forms has been the most sensitive system available. The RIA method, unfortunately, has several disadvantages, including the requirement of special equipment, trained staff, the need for extra safety measures to protect against harmful radiation, special licensing, and the regular disappearance of labeled compound by radioactive decay. The possibility of replacing the radioactive label with an enzyme label was proposed in 1968 in an article by L. E. M. Miles and C. N. Hales, entitled "Labelled Antibodies and Immunological Assay Systems," Lancet, II, 492 (1968) and Nature 219, 168 (1968), but no procedural details were provided, the article failing to offer more than the general idea, leaving it to future workers to determine the basic steps and to perform the extensive experimentation needed to establish a practical operative enzymatic immunoassay method.
While the present invention can be employed for RIA methods, it grew out of the application of EIA methods to the measurement of antigens, haptens, and antibodies to overcome the classical problems of RIA.
The pioneering work on enzyme-immunoassay (EIA) methodology (wherein one component in the immunochemical reaction is preferably insolubilized) was performed by Schuurs and coworkers, and is disclosed in a series of their U.S. Pat. Nos. 3,654,090, 3,791,932, 3,850,752, 3,839,153, 3,879,262, 4,016,043, and Re 29,169. Another line of EIA methodology is the so-called "homogenous" form which does not require separation of free and bound enzyme label, because the assay depends on the inhibition or activation of the enzyme label by antibody binding. See G. Brian Wisdom, "Enzyme-Immunoassay," Clinical Chemistry 22/8, 1243 (1976). Unfortunately, while RIA had the disadvantages of special equipment, special licenses, and highly-trained staff, its inherent sensitivity down to 10.sup.-12 gram antigen was highly desirable, especially for the detection and determination of antibodies and antigens relating to the diseases of hepatitis-B surface antigen (hereinafter referred to simply as hepatitis), rubella, and Neisseria gonorrhoeae (hereinafter referred to simply as gonorrhoea) which are difficult, if not impossible, to measure by homogenous EIA techniques because impractically large volumes of sample are required, and difficult by normal insolubilized forms of EIA techniques.
In an effort to raise the sensitivity of various marker techniques, several investigators have attempted to increase the number of markers per hapten, antigen, or antibody. With respect to FIA, John Axel Sjoquist in German Pat. No. 2,430,356 (Jan. 1, 1975) disclosed the methods for the qualitative or quantitative determination of various immunoglobulins, their Fc-fragments, or antigens or haptens (where the antigen or hapten is bound to the Fab-part of the immunoglobulin), using suitable polypeptides (such as "Protein A" obtained from Staphylococcus aureus). In German Pat. No. 2,557,419, Thomas Hirschfield disclosed FIA analytical reagents comprising (a) a reactive molecule (such as hapten, antigen, or antibody) with several reactive sites, one of which is capable of undergoing a specific reaction with the species to be analyzed, (b) a polyfunctional polymer lattice covalently bonded to the reactant molecule at a site sterically removed from the specific reaction site of the reactant molecule so as not to interfere with the specificity of the reagent; and (c) fluorescent dye molecules bonded to the polymer lattice in an amount insufficient to interfere with the specificity of the reagent. The polymer is preferably a polyethyleneimine having a molecular weight of 1200-60,000, and the reactant molecule is preferably an antibody (for antigen determination) of a ligand, such as dibenzylglyoxime (for the determination of polyvalent molecules). See also "Tagging allows molecule viewing,38 Industrial News Feb., 1977, page 23.
With respect to EIA, RIA, and FIA, William J. Dreyer in U.S. Pat. No. 3,853,987 (1974) disclosed the use a reagent consisting of microscopic carrier particle materials bearing (1) marker material--fluorescent compounds, radioactive compounds, or enzymes, or others--and (2) a coating of biological antibody for the substance (hapten, antigen, or antibody) whose assay is desired. The carrier is shown in the examples to be hydrolysed polyacrylamide resin, but can also be other acrylic acid derivatives, styrene polymers, agar, agarose, cellulose acetate, etc.
For a review of related subjects, see also Michel F. Aubert, "Critical Study of the Radioimmunological Assay for the Dosage of the Polypeptide Hormones in Plasma," J. Nuclear and Biological Medicine 13, 1-19 (1970); Robert Roberts, B. E. Sobel and C. W. Parker, "Radioimmunoassay for Creatine Kinase Isoenzymes," Science 194, 855-856 (Feb., 1977); Robert Roberts and A. Painter, "Radioimmunoassay for Carrier Creatine Kinase Isoenzymes, Biochimica Biophysica Acta 480, 521-526 (1977). Michael G. Grattain, J. M. Puttman, and T. G. Pretlow in "The Use of Glutaraldehyde-Conjugated Horseradish Peroxidase-Bovine Serum Albumin in the Visualization of Concanavalin A Binding to Tissue Sections of Human Colonic Tumor," Laboratory Investigation 35/6, 537-541 (1976) reported the method for the preparation of glutaraldehyde cross-linked horseradish peroxidase conjugates where bovine serum albumin (BSA) was used as the carrier, stating that soluble polymers of horseradish peroxidase linked to BSA will produce staining of greater intensity (without loss of specificity) than horseradish peroxidase monomers in the demonstration of the binding of concanavalin A to tissue sections.
Even after the teachings of U.S. Pat. No. 3,853,987, Ger. Pat. Nos. 2,430,356 and 2,557,419, as well as the literature above, what was needed in order to apply EIA and other non-isotopic technology to diseases tested formerly by RIA methods was a flexible, easily made, stable, and relatively inexpensive vehicle able to support several enzyme markers per antigen, anti-body, hapten, or other molecule of interest in order to improve the sensitivtiy of the immunoassays employed. Through the use of the novel diagnostic spine tool disclosed, not only are EIA and other non-isotropic immunoassays able to be applied with great sensitivity for quantitative and qualitative analysis of diseases such as hepatitis, rubella, and gonorrhea, but surprisingly the same spine tool of my invention can also be employed with other markers such as radioisotopes, fluorescent groups, lysis-initiating compounds etc. Likewise, the novel diagnostic tools of my invention can also be utilized to make even RIA more sensitive than available with the prior art.